Augmentation of limb perfusion and reversal of tissue ischemia produced by ultrasound-mediated microbubble cavitation

Abstract

Background: Ultrasound can increase tissue blood flow, in part, through the intravascular shear produced by oscillatory pressure fluctuations. We hypothesized that ultrasound-mediated increases in perfusion can be augmented by microbubble contrast agents that undergo ultrasound-mediated cavitation and sought to characterize the biological mediators.

Methods and results: Contrast ultrasound perfusion imaging of hindlimb skeletal muscle and femoral artery diameter measurement were performed in nonischemic mice after unilateral 10-minute exposure to intermittent ultrasound alone (mechanical index, 0.6 or 1.3) or ultrasound with lipid microbubbles (2×10(8) IV). Studies were also performed after inhibiting shear- or pressure-dependent vasodilator pathways, and in mice with hindlimb ischemia. Ultrasound alone produced a 2-fold increase (P<0.05) in muscle perfusion regardless of ultrasound power. Ultrasound-mediated augmentation in flow was greater with microbubbles (3- and 10-fold higher than control for mechanical index 0.6 and 1.3, respectively; P<0.05), as was femoral artery dilation. Inhibition of endothelial nitric oxide synthase attenuated flow augmentation produced by ultrasound and microbubbles by 70% (P<0.01), whereas inhibition of adenosine-A2a receptors and epoxyeicosatrienoic acids had minimal effect. Limb nitric oxide production and muscle phospho-endothelial nitric oxide synthase increased in a stepwise fashion by ultrasound and ultrasound with microbubbles. In mice with unilateral hindlimb ischemia (40%-50% reduction in flow), ultrasound (mechanical index, 1.3) with microbubbles increased perfusion by 2-fold to a degree that was greater than the control nonischemic limb.

Conclusions: Increases in muscle blood flow during high-power ultrasound are markedly amplified by the intravascular presence of microbubbles and can reverse tissue ischemia. These effects are most likely mediated by cavitation-related increases in shear and activation of endothelial nitric oxide synthase.

Keywords: microbubbles; nitric oxide; peripheral arterial disease; ultrasound.

Figure 1

(A) Peak negative acoustic pressure measurements in the lateral and elevational dimension for the two different therapeutic ultrasound conditions. Pressure measurements are color-coded according to the scale denoted in units of MPa. (B) Graphic representation of the peak negative acoustic pressure in the elevational dimension at the center of the probe, correlating to the length of muscle exposed when imaging in the short-axis plane.

Figure 2

Contrast ultrasound perfusion data from ultrasound-exposed and control contralateral hindlimbs using 2×10⁸ microbubble dosing. (A) Examples of time-intensity curves from the adductor muscle group obtained after a destructive pulse sequence in a leg after exposure to ultrasound (MI 1.3) in the presence of MBs and in the contralateral control leg. (B) Contrast ultrasound frames obtained from select intervals from the curves in panel A (T₀=immediate post-destruction). (C and D) Dot plots with lines representing mean (±SD) of skeletal muscle microvascular blood flux rate (β) and microvascular blood flow (MBF) after each of the experimental conditions for the ultrasound exposed and contralateral control limb. MB, microbubble; MI, mechanical index. *p<0.05 versus contralateral control limb; †p=0.05 versus contralateral control limb; all tests were made by non-parametric analysis.

Figure 3

Femoral artery dilation with ultrasound. (A) Box-whisker plots showing median (horizontal line), intequartile range (box), and 5–95^th percentile (whiskers) for the percent change in femoral artery diameter measured after each of the experimental conditions for the ultrasound exposed and contralateral control limb. (B) Example of a femoral artery at baseline and after exposure to ultrasound (MI 1.3) and MBs. *p<0.05 versus contralateral control limb by paired analysis; †p<0.05 vs. baseline; all tests were made by non-parametric analysis.

Figure 4

Mean (±SEM) microvascular flux rate (A), microvascular blood flow (MBF) (B) and ratio of MBF in the ischemic to contralateral limb (C) measured in mice with chronic hindlimb ischemia (n=12). *p<0.05 vs. control limb; †p<0.05 vs. baseline.

Figure 5

Contrast ultrasound perfusion data in the presence of inhibitors to vasoactive compounds (n=5 for each condition). Data were obtained after 10 min exposure to intermittent ultrasound (MI 1.3) with MBs alone or after inhibition of NOS (L-NAME), adenosine-A2a receptor signal (ZM241385), or EET signaling (EEZE). Mean (±SEM) data control and ultrasound exposed leg are shown for (A) microvascular blood flux rate (β) and (B) microvascular blood flow (MBF). *p<0.01 versus contralateral control leg.

Figure 6

(A) Time dependent increases in fluorescent intensity from the NO indicator DAF-2 from cultured endothelial cells exposed to ultrasound with and without microbubbles (MBs), and from non-exposed time-controlled cells. (B) Phosphorylated eNOS by ELISA from control muscle and muscle tissue within the imaging sector in muscle exposed to ultrasound with and without MB injection.

Figure 7

(A) Intramuscular NO production measured by indwelling electrochemical probe after initiating intermittent US alone (n=6), intermittent US with MBs (n=6), and intermittent US with MBs and L-NAME (n=2). Data are normalized to baseline values. (B) Intramuscular electrochemical probe detection of NO production after initiating US with MBs where measurements were continued for 10 min after cessation of US (n=4). (C) Example of continuous time-dependent NO detection in a limb exposed to ultrasound (MI 1.3) with MBs and the contralateral control limb. Statistical analysis performed with parametric tests.

Figure 8

Frequency-amplitude histograms from passive cavitation detection from microbubbles exposed to ultrasound at MI 0.6 or 1.3. Data are averaged from 250 measurements.